Taku Yamaguchi

Role of the PLC-related, catalytically inactive protein p130 in GABAA receptor function

The protein p130 was isolated from rat brain as an inositol 1,4,5‐trisphosphate‐binding protein with a domain organization similar to that of phospholipase C‐δ1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for γ‐aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABAA receptor‐associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABAA receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the γ2 subunit of the GABAA receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA‐induced Cl− current by Zn2+ or diazepam, both of which act at GABAA receptors containing γ subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABAA receptors, especially in response to the agents acting on a γ2 subunit.

Phosholipase C-Related Inactive Protein Is Involved in Trafficking of γ2 Subunit-Containing GABAA Receptors to the Cell Surface

The subunit composition of GABAA receptors is known to be associated with distinct physiological and pharmacological properties. Previous studies that used phospholipase C-related inactive protein type 1 knock-out (PRIP-1 KO) mice revealed that PRIP-1 is involved in the assembly and/or the trafficking of γ2 subunit-containing GABAA receptors. There are two PRIP genes in mammals; thus the roles of PRIP-1 might be compensated partly by those of PRIP-2 in PRIP-1 KO mice. Here we used PRIP-1 and PRIP-2 double knock-out (PRIP-DKO) mice and examined the roles for PRIP in regulating the trafficking of GABAA receptors. Consistent with previous results, sensitivity to diazepam was reduced in electrophysiological and behavioral analyses of PRIP-DKO mice, suggesting an alteration of γ2 subunit-containing GABAA receptors. The surface numbers of diazepam binding sites (α/γ2 subunits) assessed by [3H]flumazenil binding were reduced in the PRIP-DKO mice as compared with those of wild-type mice, whereas the cell surface GABA binding sites (α/β subunits, assessed by [3H]muscimol binding) were increased in PRIP-DKO mice. The association between GABAA receptors and GABAA receptor-associated protein (GABARAP) was reduced significantly in PRIP-DKO neurons. Disruption of the direct interaction between PRIP and GABAA receptor β subunits via the use of a peptide corresponding to the PRIP-1 binding site reduced the cell surface expression of γ2 subunit-containing GABAA receptors in cultured cell lines and neurons. These results suggest that PRIP is implicated in the trafficking of γ2 subunit-containing GABAA receptors to the cell surface, probably by acting as a bridging molecule between GABARAP and the receptors.

Endogenous cannabinoid, 2-arachidonoylglycerol, attenuates naloxone-precipitated withdrawal signs in morphine-dependent mice.

In the present study, we examined the effects of endogenous ligand 2-arachidonoylglycerol (2-AG) on naloxone-precipitated withdrawal in morphine-dependent mice, in comparison with that of two cannabinoid agonists, an ingredient of Cannabis sativa Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and the synthetic cannabinoid CB1 receptor agonist HU-210. 2-AG at a dose of 10 microg per mouse (i.c.v.) significantly inhibited both jumping and forepaw tremor as signs of withdrawal following naloxone challenge in morphine-dependent mice. Furthermore, both Delta(8)-THC and HU-210 significantly attenuated these symptoms of withdrawal in morphine-dependent mice. Therefore, it is suggested that inactivation of the endogenous cannabinoid system is related to the induction of withdrawal syndrome in morphine-dependent mice. Moreover, hyperlocomotor activity in morphine-dependent mice was markedly increased by Delta(8)-THC 10 mg/kg, which had no effect in naive mice. This finding suggested that in morphine dependence, upregulation of cannabinoid CB1 receptors occurred. Non-psychoactive CB1 receptor agonists or accelerators of endocannabinoid synthesis may be potential as therapeutic drugs for opiate withdrawal symptoms.

Activation of the β-Adrenoceptor–Protein Kinase A Signaling Pathway within the Ventral Bed Nucleus of the Stria Terminalis Mediates the Negative Affective Component of Pain in Rats

Pain is an unpleasant sensory and emotional experience. The neural systems underlying the sensory component of pain have been studied extensively, but we are only beginning to understand those underlying its affective component. The bed nucleus of the stria terminalis (BNST) has been implicated in stress responses and negative affective states, such as anxiety, fear, and aversion. Recently, we demonstrated the crucial role of the BNST in the negative affective component of pain using the conditioned place aversion (CPA) test. In the present study, we investigated the involvement of the β-adrenoceptor–protein kinase A (PKA) signaling pathway within the BNST, in particular, within the ventral part of the BNST (vBNST), in pain-induced aversion in male Sprague Dawley rats. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST were significantly increased by intraplantar formalin injection. Using the CPA test, we found that intra-vBNST injection of timolol, a β-adrenoceptor antagonist, dose-dependently attenuated the intraplantar-formalin-induced CPA (F-CPA) without reducing nociceptive behaviors. Experiments with subtype-selective antagonists demonstrated the essential role of β2-adrenoceptors in F-CPA. Intra-vBNST injection of isoproterenol, a β-adrenoceptor agonist, dose-dependently produced CPA even in the absence of noxious stimulation. This isoproterenol-induced CPA was reversed by the coinjection of Rp-cyclic adenosine monophosphorothioate (Rp-cAMPS), a selective PKA inhibitor. Furthermore, intra-vBNST injection of Rp-cAMPS dose-dependently attenuated the F-CPA. Together, these results suggest that PKA activation within the vBNST via the enhancement of β-adrenergic transmission is important for the negative affective component of pain.

Recurrent Trigeminal Neuralgia After Microvascular Decompression Using an Interposing Technique. Teflon Felt Adhesion and the Sling Retraction Technique

Summary¶ The causes of recurrence after microvascular decompression for trigeminal neuralgia and the results of re-operations were studied in 6 cases. Eighty-two patients with trigeminal neuralgia were operated on through microvascular decompression using the technique of interposing Teflon felt between the offending artery and the pons and/or nerve. Recurrence occurred in 14 cases (17.1%) and re-operations were carried out in 6 severe cases at which time the sling retraction technique was used. At the second operation, the adhesion of the interposed Teflon felt was found at the trigeminal nerve in all cases and the adhesions were the main cause of recurrence. The Teflon felt was dissected from the nerve, and the sling of the Teflon felt adhering to the offending arteries was fixed to the tentorium in order to transpose the arteries and avoid re-adhesion. All cases resulted in an excellent relief from pain and experienced no pain for at least 2 years. The intra-operative findings of our cases indicated that the microvascular decompression using the interposing technique may result in adhesion of the prosthesis to the nerve and thus eventually lead to recurrence. Our surgical experience also suggests that such recurrent cases should be re-operated on using the sling retraction technique instead of the interposing technique, even for the first microvascular decompression procedure.

Involvement of arachidonic acid cascade in working memory impairment induced by interleukin-1 beta

The aim of the present study is to clarify the possible relevance of the arachidonic acid cascade to working memory in rats, by using a three-panel runway apparatus. Interleukin-1 beta, injected bilaterally into the dorsal hippocampus at a dose of 100 ng/side, significantly impaired working memory, and this impairment was attenuated by pretreatment with 10 mg/kg (s.c.) of diclofenac, a cyclooxygenase inhibitor. Working memory was also impaired in rats administered a bilateral intrahippocampal injection of prostaglandin E2, in a dose-dependent manner at 0.01-1 microg/side. Furthermore, an injection of 100 ng/side of interleukin-1 beta significantly increased production of prostaglandin E2 (580 +/- 32 pg to 1142 +/- 101 pg/100 mg wet tissue) in the hippocampus. Taken together, these findings suggest that the activation of the arachidonic acid cascade was causative of the working memory impairment induced by interleukin-1 beta.The aim of the present study is to clarify the possible relevance of the arachidonic acid cascade to working memory in rats, by using a three-panel runway apparatus. Interleukin-1 beta, injected bilaterally into the dorsal hippocampus at a dose of 100 ng/side, significantly impaired working memory, and this impairment was attenuated by pretreatment with 10 mg/kg (s.c.) of diclofenac, a cyclooxygenase inhibitor. Working memory was also impaired in rats administered a bilateral intrahippocampal injection of prostaglandin E2, in a dose-dependent manner at 0.01-1 μg/side. Furthermore, an injection of 100 ng/side of interleukin-1 beta significantly increased production of prostaglandin E2 (580±32 pg to 1142±101 pg/100 mg wet tissue) in the hippocampus. Taken together, these findings suggest that the activation of the arachidonic acid cascade was causative of the working memory impairment induced by interleukin-1 beta.

Role of enhanced noradrenergic transmission within the ventral bed nucleus of the stria terminalis in visceral pain-induced aversion in rats

Pain is an unpleasant sensory and emotional experience. We demonstrated the crucial role of the bed nucleus of the stria terminalis (BNST) in the negative affective component of somatic and visceral pain induced by intraplantar formalin and intraperitoneal acetic acid injections, respectively, in rats. Recently, we reported the involvement of enhanced noradrenergic transmission via beta-adrenoceptors within the ventral BNST (vBNST) in formalin-induced aversion. Here, we examined the role of intra-vBNST noradrenergic transmission in the negative affective component of visceral pain induced by intraperitoneal acetic acid injection. In vivo microdialysis showed that extracellular noradrenaline levels within the vBNST significantly increased after intraperitoneal acetic acid injection. Using a conditioned place aversion (CPA) test, we found that intra-vBNST injection of timolol, a beta-adrenoceptor antagonist, dose-dependently attenuated the acetic acid-induced CPA without reducing nociceptive behaviors. These results suggest that enhanced noradrenergic transmission via beta-adrenoceptors within the vBNST plays a pivotal role in the negative affective, but not sensory, component of visceral pain.

The effects of serotonin and/or noradrenaline reuptake inhibitors on impulsive-like action assessed by the three-choice serial reaction time task: a simple and valid model of impulsive action using rats.

Impulsivity is a pathological symptom in several psychiatric disorders, underscoring the need for animal models of impulsive action to develop a brief screening method for novel therapeutic agents of impulsive action. The aims of this study were (i) to evaluate whether the three-choice serial reaction time task (3-CSRTT), a simple version of the five-choice serial reaction time task (5-CSRTT), is appropriate for brief assessment of impulsive-like action and (ii) to examine the effects of fluvoxamine, a selective serotonin reuptake inhibitor, and milnacipran, a serotonin/noradrenaline reuptake inhibitor, on impulsive-like action using the 3-CSRTT. After training in the 3-CSRTT, rats were administered nicotine (0, 0.1, 0.2, and 0.4 mg/kg, salt, subcutaneously), atomoxetine [0, 0.01, 0.1, and, 1.0 mg/kg, intraperitoneally (i.p.)], fluvoxamine (0, 2, 4, and 8 mg/kg, i.p.), or milnacipran (0, 3, and 10 mg/kg, i.p.). The training time for the 3-CSRTT was significantly shorter than that for the 5-CSRTT. Nicotine increased, whereas atomoxetine decreased the number of premature responses, an index of impulsive-like action, which is consistent with earlier studies. Milnacipran, but not fluvoxamine, dose-dependently decreased premature responses. These results indicate that the 3-CSRTT could provide an appropriate and simpler rodent model of impulsive-like action and that milnacipran could have some beneficial effects on impulsivity-related disorders.

Corticotropin releasing factor enhances attentional function as assessed by the five-choice serial reaction time task in rats

Previous studies have shown that psychological stress affects attentional function, and corticotropin releasing factor (CRF) is closely related to stress responses. In the present study, we examined the effect of CRF on attentional function using a five-choice serial reaction time task (5-CSRTT) in rats. Accuracy in the 5-CSRTT was used as the index of attentional function. Human/rat CRF was intracerebroventricularly injected (0, 0.01, 0.1, or 1 microg/5 microl saline) 20 min before the beginning of the test trial in male Lister hooded rats that generally show high performance in the 5-CSRTT. As a result, 0.1 microg of CRF, but not other doses of CRF, increased accuracy in the 5-CSRTT. However, 0.1 microg of CRF did not affect impulsivity, motivation/appetite, perseverative tendency, or motor function. Even when Wistar rats, which generally show poor performance in the 5-CSRTT were used, 0.1 microg of CRF increased accuracy in the 5-CSRTT. However, the effect of CRF was not significantly attenuated by intracerebroventricular injection of the CRF(1) receptor antagonist antalarmin (5 microg). These results showed that CRF selectively enhances attentional function regardless of baseline attentional performance and rat strain, but this effect may be due to the pathways other than CRF(1) receptors. The present results suggest that CRF is involved in stress-related changes of attention and indicate that moderate stress, but not severe stress, may enhance attentional performance. Furthermore, these findings also indicate that the CRF-related substance could be a target for the development of an agent to improve attentional function.

Early postnatal stress alters the extinction of context-dependent conditioned fear in adult rats

Fear extinction is hypothesized to be a learning process based on a new inhibitory memory. The present study was conducted to elucidate the effect of early postnatal stress on the extinction of context-dependent fear memory in adult rats, with a focus on the serotonergic system. Extinction was estimated by the expression of freezing behavior during repeated extinction trials (i.e., repeated exposure to contextual fear conditioning) on consecutive days. The decrease in fear expression was attenuated in adult rats that had been subjected to footshock (FS) at the third postnatal week (3wFS), but not in those exposed to footshock at the second postnatal week (2wFS). The decreased attenuation of freezing behavior observed in 3wFS was abolished by repeated treatment with the partial N-methyl-D-aspartate receptor agonist D-cycloserine (15 mg/kg, i.p., for 4 days), which has been shown to facilitate cue-dependent extinction. Repeated treatment with the serotonin 5-hydroxytryptamine-1A (5-HT(1A)) receptor agonist tandospirone (1 mg/kg, i.p., for 4 days) prevented the expression of freezing behavior in 3wFS, whereas diazepam treatment (1 mg/kg, i.p., for 4 days) in 3wFS did not. These results suggest that exposure to early postnatal stress at the third week is responsible for attenuating extinction of contextual fear conditioning and is mediated by a serotonergic 5-HT(1A) receptor mechanism. In other words, exposure to traumatic events during the early postnatal period might precipitate long-lasting alterations in synaptic function that underlie extinction processes of context-dependent fear memory.